The hallmark of myelinated axons is their organization into molecularly distinct domains, a pre-requisite for the rapid propagation of action potentials. The paranodal domains establish the axo-glial septate junctions (AGSJs) through interactions involving axonal Contactin-associated protein (Caspr), Contactin (Cont), and glial Neurofascin NF155. The nodal domain is organized by neuronal NF186, voltage gated sodium (Nav) channels, and Ankyrin G (AnkG), a cytoskeletal adaptor protein. We showed that loss of Caspr and NF155 results in loss of AGSJs, mislocalization of the juxtaparanodal proteins, disorganization of the paranodal axonal cytoskeleton, and degeneration of myelinated axons, but nodal organization remains relatively unaffected. Recently, we showed that Band 4.1B, a paranodal/ juxtaparanodal cytoskeletal protein, is essential for the stability of AGSJs and juxtaparanodal organization. We show here that Whirlin, another cytoskeletal protein, is required for paranodal compaction and cytoskeletal stability. In further studies, we demonstrated that loss of nodal NF186 abolished clustering of Nav channels and AnkG at the nodes, allowing the flanking paranodal AGSJs to invade the nodal space. Most importantly, we show here that in vivo loss of AnkG does not abolish node formation, but may affect nodal stability. While significant advancements have been made regarding the composition and organization of axonal domains, there still remain fundamental questions regarding how the transmembrane components at these domains interact with local cytoskeleton to initiate domain organization, and to ensure long-term stability and maintenance of axonal architecture. Based on our published and preliminary studies, our central hypothesis is that the transmembrane components and local axonal cytoskeleton are critical for axonal domain organization, their stability and function. We will use genetic, molecular and cell biological methods to determine the specific role of paranodes, nodes and their associated cytoskeletal proteins in domain stabilization and maintenance, and the efficacy of restoration of key axonal domains by re-expression of NF155 and NF186 in progressively weak adult mouse mutants. We will accomplish our goals in the following specific aims: (1) What are the consequences of loss of cytoskeletal scaffolding proteins and loss of NF155 during adult life on the maintenance and function of paranodal AGSJs? (2) What are the consequences of loss of nodal cytoskeletal proteins and loss of NF186 during adult life on the stability and function of nodes in myelinated axons? and (3) Are adult myelinated axons with extended periods of disorganized domain structure able to re-organize axonal domains to restore nerve conduction? Collectively, our studies will provide insights that bear directly on the mechanisms by which axonal domains are formed and maintained, and how these structures can be reorganized and nerve function restored. In the future, these studies will advance our understanding of how demyelinating diseases, such as multiple sclerosis (MS) lead to axonal domain disorganization and guide the development of therapeutic interventions.